Stimulation of pancreatic islets with D-glucose induces phospholipid hydrolysis and accumulation of non-esterified arachidonic acid, as demonstrated by isotope dilution mass spectrometry. Similar events occur in HIT insulinoma cells, and the accumulation of arachidonate may participate in the glucose-induced in beta cell (Ca2+]. The identity of the phospholipase enzyme which catalyzes glucose-induced hydrolysis of beta cell phospholipids is not known. ATFMK, an analogue of AA, inhibits an 85 kDa cytosolic phospholipase A2 enzyme. Exposure of HIT insulinoma cells to ATFMK induced a delayed, sustained, and irreversible increase in cytosolic [Ca2+] that required extracellular Ca2+, and a concentration-dependent inhibition of depolarization-induced increases in cytosolic [Ca2+], prior to onset of the delayed response to AFTMK. These results suggest a disruptive effect of ATFMK on calcium mobilization which may contribute, in part, to its effects on insulin secretion from (-ce lls.